REVIEWpublished: 13 October 2016

doi: 10.3389/fmicb.2016.01601

Frontiers in Microbiology | www.frontiersin.org 1 October 2016 | Volume 7 | Article 1601

Edited by:

Dongsheng Zhou,

Beijing Institute of Microbiology and

Epidemiology, China

Reviewed by:

Baolin Sun,

University of Science and Technology

of China, China

Lefu Lan,

Shanghai Institute of Materia Medica

(CAS), China

Fangyou Yu,

First Affiliated Hospital of Wenzhou

Medical University, China

*Correspondence:

Xiancai Rao

raoxiancai@126.com

Specialty section:

This article was submitted to

Antimicrobials, Resistance and

Chemotherapy,

a section of the journal

Frontiers in Microbiology

Received: 22 July 2016

Accepted: 26 September 2016

Published: 13 October 2016

Citation:

Hu Q, Peng H and Rao X (2016)

Molecular Events for Promotion of

Vancomycin Resistance in

Vancomycin Intermediate

Staphylococcus aureus.

Front. Microbiol. 7:1601.

doi: 10.3389/fmicb.2016.01601

Molecular Events for Promotion ofVancomycin Resistance inVancomycin IntermediateStaphylococcus aureus

Qiwen Hu, Huagang Peng and Xiancai Rao*

Department of Microbiology, College of Basic Medical Sciences, Third Military Medical University, Chongqing, China

Vancomycin has been used as the last resort in the clinical treatment of serious

Staphylococcus aureus infections. Vancomycin-intermediate S. aureus (VISA) was

discovered almost two decades ago. Aside from the vancomycin-intermediate

phenotype, VISA strains from the clinic or laboratory exhibited common characteristics,

such as thickened cell walls, reduced autolysis, and attenuated virulence. However, the

genetic mechanisms responsible for the reduced vancomycin susceptibility in VISA are

varied. The comparative genomics of vancomycin-susceptible S. aureus (VSSA)/VISA

pairs showed diverse genetic mutations in VISA; only a small number of these mutations

have been experimentally verified. To connect the diversified genotypes and common

phenotypes in VISA, we reviewed the genetic alterations in the relative determinants,

including mutations in the vraTSR, graSR, walKR, stk1/stp1, rpoB, clpP, and cmk

genes. Especially, we analyzed the mechanism through which diverse mutations mediate

vancomycin resistance. We propose a unified model that integrates diverse gene

functions and complex biochemical processes in VISA upon the action of vancomycin.

Keywords: vancomycin intermediate Staphylococcus aureus, vancomycin, genetic mechanisms, genotypes,

molecular events

INTRODUCTION

Staphylococcus aureus is a successful human pathogen because of its metabolic versatility andits ability to adapt to host defensive stress (Didelot et al., 2016). This pathogen can cause mildinfections and life-threatening diseases, including skin and soft tissue infections, bacteremia,pneumonia, endocarditis, sepsis, and toxic shock syndrome (Dayan et al., 2016). Unfortunately,a licensed vaccine is unavailable for S. aureus infections. The optimal choice for treatment ofS. aureus infections is the employment of antibiotics. However, antimicrobial resistance in S.aureus has become a major public health threat. The first antibiotic penicillin was discovered byAlexander Fleming in 1928 based on the susceptibility of S. aureus; subsequently, penicillin wasclinically applied in a large scale during the early 1940s. After several years, the penicillin-resistantS. aureus (PRSA) was characterized in hospitals in the mid-1940s. PRSA strains usually carry aplasmid-encoded penicillinase, which can hydrolyze the β-lactam ring of penicillin to inactivate itsantimicrobial activity. PRSA strains become pandemic by the early 1950s and were significantlycontrolled by the introduction of β-lactamase-resistant methicillin into clinic in 1959. However,the first methicillin-resistant S. aureus (MRSA) strain was quickly generated and isolated in 1961then spreaded globally. MRSA strains are inherently resistant to virtually all β-lactam antibiotics,

Hu et al. Molecular Events in VISA

including penicillins, cephalosporins, and carbapenems. Theemergence of MRSA resistance is the horizontal gene transfer ofthe mecA gene, which encodes an alternative penicillin bindingprotein 2a (PBP2a) with low-affinity to β-lactam antibiotics.With complicated evolution, MRSA has become a so-called“superbug” that has acquired resistance to multiple drugs, frompenicillin/methicillin to quinolone and vancomycin (Nordmannet al., 2007).

Vancomycin is a cationic glycopeptide antibiotic derived fromthe organism Amycolatopsis orientalis (previously known asStreptomyces orientalis or Nocardia orientalis). Vancomycin wasfirst discovered by Edmund Kornfield (working at Eli Lilly) in1953 and approved by the Food and Drug Administration ofthe United States in 1958 because of the rapid development ofpenicillin resistance by staphylococci (Levine, 2006). However,due to the ototoxicity and nephrotoxicity caused by thepresence of impurities and the development of other effectiveantibiotics, vancomycin was relegated to a second-line antibioticin the 1960s and 1970s (Moellering, 2006). Vancomycin killsbacteria by binding to the C-teminal D-Ala–D-Ala residuesof the peptidoglycan precursor lipid II in the cytoplasmicmembrane to form a stable, non-covalent complex, whichprevents the use of the precursor for cell wall synthesis (Figure 1).The steric hindrance imparted by vancomycin may furtherinhibit the glycosyltransferase and transpeptidase activitiesof penicillin-binding proteins (PBPs) (Kahne et al., 2005).Clinically, vancomycin is used for S. aureus infections andfor infections caused by Streptococcus pneumoniae, Clostridiumdifficile, Enterococcus species, and so on. The increasing burden ofMRSA and other Gram-positive bacterial infections in hospitalsled to the increasing use of vancomycin worldwide since the1980s (Levine, 2006). From 1980s to present, vancomycin isone of the last remaing antibiotics to which most of the MRSAand other multiple drug-resistant Gram-positive bacteria werestill reliably susceptible. Moreover, vancomycin is used to treatosteomyelitis, bacteremia, and endocarditis empirically or whenMRSA is deemed a possible cause (Rubinstein and Keynan,2014). However, vancomycin-resistant Enterococcus (VRE) wasfirst reported in 1986 in Europe then in the USA in 1987(Murray, 2000) (Figure 2). S. aureus clinical isolates with reducedvancomycin susceptibility, such as the vancomycin-intermediateresistance S. aureus (VISA) strain Mu50 (MIC = 8 µg/mL)and the heterogeneous VISA (hVISA) strain Mu3 (MIC =

3 µg/mL), were first reported in Japan in 1997 (Hiramatsuet al., 1997a,b) then reported globally. VISA usually exhibitsa low level of resistance, as defined by a vancomycin MICfrom 4 to 8 µg/mL, although laboratory-derived VISA strainswith vancomycin MICs of 32–100 µg/mL were achieved byin vitro mutagenesis (Berscheid et al., 2014; Ishii et al., 2015).Furthermore, the first vancomycin-resistant S. aureus (VRSA)isolate MI-1, with an MIC of 128 µg/mL, was recovered in 2002from the foot wound of a diabetic patient who had receivedlong-term vancomycin therapy and also had a VRE isolate.

VRSA is a rare, multidrug-resistant bacterial strain of publichealth concern. Since its emergence in the USA in 2002, 36 VRSAstrains have been reported, 14 from the USA (Limbago et al.,2014; Walters et al., 2015), 16 from India, 3 from Iran, 1 from

FIGURE 1 | The mode of action of vancomycin in S. aureus. By binding

to the C-teminal D-Ala-D-Ala residues of the pentapeptide, vancomycin

inhibits the cross bridge formation between pentapeptide and pentaglycine.

GlcNAc, N-acetylglucosamine; MurNAc, N-Acetylmuramic acid.

Pakistan (Moravvej et al., 2013), 1 form Portugal (Melo-Cristinoet al., 2013), and 1 from Guatemala (Antony, 2014). VRSA ariseswhen vancomycin resistance genes (e.g., the vanA operon, whichcodes for enzymes that result in modification or elimination ofthe vancomycin-binding site) from VRE are transferred to S.aureus. The vanA gene cluster is often located in the plasmid-borne transposon Tn1546, which can transfer from vancomycin-resistant Enterococcus faecalis to S. aureus and produce a VRSAisolate (Zhu et al., 2013). The mechanism for VRSA resistance iswell-characterized. The coded product enables VRSA to replacethe D-Ala–D-Ala terminal dipeptide with D-Ala–D-Lac dipeptide,thereby altering the binding target of vancomycin and oftenmediating high level resistance to vancomycin. Although VRSAmay have been underestimated (Moravvej et al., 2013), thelimited cases of VRSA suggest that vanA-mediated vancomycinresistance is significant but has not evolved or quickly spread;this trend is probably caused by the high fitness cost imparted byvanA-type transposon (Foucault et al., 2009). By contrast, VISAor hVISA isolates have been characterized from many countriesaround the world, including United States, France, Australia,Scotland, Brazil, Japan, South Korea, China, and other countries(Howden et al., 2010; Zhang et al., 2013). A retrospectivestudy found that hVISA emerged before the introduction ofvancomycin in Japan in 1990 (Yamakawa et al., 2012). Twoother retrospective studies also revealed that the emergence ofhVISA or VISA isolates in the USA or in Europe can be datedback to the mid or late 1980s (Rybak et al., 2005; Robert et al.,2006). Pulsed field gel electrophoresis and multilocus sequencetyping found that VISA strains were not clonal. Most VISA werereported to evolve from hospital associated MRSA; mainly fromclonal complex 5 or 8, in particular ST5 and ST239, however,the community associated MRSA clone USA300-derived andmethicillin-susceptible S. aureus (MSSA)-derived VISA are alsoreported (Pillai et al., 2009; Gardete et al., 2012). In recent years,many studies also revealed that there is a vancomycin MIC creepinmany countries but not consistently observed in different areas(Sader et al., 2009; Reynolds et al., 2012; Zhuo et al., 2013). ThehVISA strains account for up to 26% of MRSA isolates in Japan(Schwaber et al., 2003), while in an Australian institute, the rate of

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Hu et al. Molecular Events in VISA

FIGURE 2 | Timeline indicates the year in which an event occurred or was reported. The increased use of vancomycin in the USA, France, Italy, Germany, the

United Kingdom, and the Netherlands was shown as tons of vancomycin in the y-axis, which was modified from reference Levine (2006).

hVISA is almost 50% (Horne et al., 2009). The high rate of hVISApaved the way for the evolution of VISA.

Aside from the vancomycin-intermediate phenotype, VISAstrains exhibit common characteristics, such as thickened cellwalls, reduced autolysis, and attenuated virulence (Howdenet al., 2011). Understanding the molecular mechanisms forreduced vancomycin susceptibility in VISA has been rapidlymoved forward by whole genome sequencing of vancomycin-susceptible S. aureus (VSSA)/VISA pairs or series (Howdenet al., 2011). The mechanisms underlying VISA resistance maybe complicated; diverse mutations occurred in VISA have beenidentified, but only a small number of mutations in several geneshave been experimentally verified. In this review, we summarizethe phenotypic features associated with VISA and thoroughlycatalog the diverse mutations in VISA, especially those thathave been experimentally characterized. Specifically, we reviewedand analyzed four categoris of genetic mutations occurred inVISA, including mutations in cell wall synthesis, hydrolysis, orremodeling genes, in metabolic genes, in transcription regulatorygenes, and in post-translational modification genes. Based onthese analyses, we connected the genetic mutations in differentgenes, biochemical functions of the cognate proteins, andkey metabolic pathways that contribute to the promotion ofvancomycin resistance in VISA.

COMMON FEATURES OF VISA

VISA exhibits several common phenotypic changes comparedwith VSSA; these features include increased cell wall thickness,reduced autolysis, decreased activity of the staphylococcal globalregulator Agr, reduced lysostaphin susceptibility, and changes incell wall teichoic acids (Nannini et al., 2010).

Thickened Cell Walls with LowCross-LinkingBacterial cell walls play important roles in the protection ofviable cells, as well as the infectivity and pathogenicity ofGram-positive and Gram-negative bacteria. Staphylococcal cell

walls are composed of murein, teichoic acids, and wall-associatedsurface proteins, with a thickness of ≈20 nm (Dmitriev et al.,2004). The stress-bearing murein of S. aureus consists of glycanstrands, which are composed of N-acetylglucosamine (GlcNAc)and N-acetylmuramic acid (MurNAc) residues that form β-(1–4)-linked disaccharide repeating units. The carboxyl group ofeach MurNAc residue of the murein is amidated by the stempentapeptide L-Ala–D-Gln–L-Lys–D-Ala–D-Ala; a pentaglycinebridge branches with the ε-amino group of the L-Lys residueof pentapeptide and the carboxyl group on the D-Ala terminusof the other pentapeptide to provide a high degree of mureincross-linking. The process of murein cross-linking is catalyzed byPBP2/PBP4 proteins.

Mu50 was the first VISA isolated from the purulent dischargeat the sternal incision site of a 4 month-old male infant whounderwent heart surgery for pulmonary atresia; its cell wallappeared to be twice as thick as that of control strains under anelectron microscope (Hiramatsu et al., 1997b). The thickness ofthe cell wall was associated with the vancomycin resistance inVISA. Cui et al. (2003) demonstrated that cell wall thickening is acommon feature of VISA isolates. Although the general principleof murein structural organization seems simple, the muropeptidecomposition of the staphylococcal cell wall appears very complexbecause the catalysis of murein cross-linking with PBPs is dose-dependent and often produces a distinctive degree of mureincross-linking. Hanaki et al. (1998) found that the incorporation of14C-lablled GlcNAc into the cell wall and the intracellular mureinmonomer precursor was increased in Mu50; the activated cellwall synthesis and the resulting thickened cell walls might be aprerequisite for vancomycin resistance in VISA. Sieradzki andTomasz (2003) revealed that VISA isolates in the JH serial strainsproduced thickened cell walls and upregulated the PBP2 proteinlevel, similar to a Chinese VISA XN108 (Zhang et al., 2013).However, the protein level of PBP4was drastically reduced, whichdecreased the cross-linking of VISA cell walls.

The thickened cell wall and decreased murein cross-linkingwere proposed to be significant for vancomycin resistance inVISA. The actual target of vancomycin is the free D-Ala–D-Ala

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Hu et al. Molecular Events in VISA

terminal of the pentapeptide. The last D-Ala should behydrolyzed by PBP4, and the pentaglycine of a murein monomercould be branched by PBP2 to finish the cross-linking. Thecell wall of S. aureus usually has ≈20% of its peptidoglycancomponents that are not crosslinked, which produced anappropriate amount of free D-Ala–D-Ala residues in the cellwall. The thickened cell wall of a VISA usually has decreasedcross-linking in its peptidoglycan, which will increase the amountof free D-Ala–D-Ala residues that allow the “capture” of morevancomycin molecules to protect VISA from the antibiotic(Sieradzki and Tomasz, 2003). Furthermore, the Gln-non-amidated murein monomer is increased in VISA, which is apoor substrate for PBPs but has a greater binding affinity forvancomycin than Gln-amidated muropeptide (Cui et al., 2000).Vancomycin has to pass through the peptidoglycan layers toreach the lipid II-bound muropeptide, which accumulates inthe division septum. Thus, most vancomycin molecules mightbe trapped by the free D-Ala–D-Ala residues in the thickenedcell walls of VISA. The cell wall thickness and vancomycinresistance are strongly correlated (Cui et al., 2000). Fluorescencemicroscopy shows that VISA strains can trap more vancomycinmolecules than VSSA strains, thus less vancomycin moleculescan arrive at the active cell wall synthesis site (Pereira et al.,2007). The thickened cell wall of Mu50 consumes more than2.8 times of vancomycin than VSSA (Cui et al., 2000). Afterbinding with vancomycin molecules, the structure of the outerlayers of VISA thickened peptidoglycan is more compact toprevent further penetration of vancomycin molecules. This so-called “vancomycin clogging effect” might also contribute tovancomycin resistance in VISA (Cui et al., 2006).

Decreased AutolysisAutolysis occurs in a wide variety of bacteria and is catalyzedby their own autolytic enzymes to cleave specific bonds inthe bacterial cell wall peptidoglycan. In S. aureus, severalmuramidases and amidases, such as lysostaphin, AltA, andLytM, participate in autolysis. The autolytic activity could bedetermined by whole-cell assays in 0.05M Tris–HCl (pH 7.2)containing 0.05% Triton X-100 or purified cell wall assays toretain the autolytic activities of the crude cell walls (Utaidaet al., 2006). VISA strains often express reduced autolytic activityvs. that of VSSA (Howden et al., 2010). Mu50 was shown tobe deficient in autolytic activity when whole-cell assays wereconducted (Utaida et al., 2006). Correspondingly, the expressionlevel of autolysins was down-regulated in VISA. The decreasedautolysis in VISA may contribute to the cell wall thickening,thereby preventing vancomycin from reaching its action site.

Decreased VirulenceAn S. aureus strain may suffer severe fitness cost upondeveloping resistance to certain antibiotics, such as resistanceto ciprofloxacin or gentamicin; this fitness cost could be adisadvantage for its pathogenesis or clonal spreading in a givenregion (Shang et al., 2016). Several animal models were used toevaluate the virulence of VISA. In Galleria mellonella, a modelinsect, clinical VISA isolates have reduced virulence comparedwith their parental strains (Peleg et al., 2009; Howden et al.,

2011). Furthermore, a VISA isolate showed reduced infectivityand rapid blood clearance compared with its parental strain ina rat endocarditis model (Majcherczyk et al., 2008). AnotherVISA isolate had attenuated virulence and lost the capacityto generate liver abscesses and tissue necrosis in a mousesepsis model (Cameron et al., 2012). S. aureus produces tensof virulence factors for pathogenesis; VISA transcriptomic datawere generally consistent with decreased virulence (Gardete et al.,2012). Hattangady et al. (2015) demonstrated that genes encodingsecreted proteases and hemolysins were generally downregulatedin two VISA strains vs. their progenitor VSSA strains. A VISAstrain named SG-R, belonging to the MRSA clone USA300,showed massive downregulation in the transcription of virulencedeterminants, including seven of the major virulence genes of S.aureus (Gardete et al., 2012). Importantly, the transcription levelof a virulence gene was restored when the vancomycin-resistantisolate SG-R was phenotypically “converted” to vancomycinsusceptibility by the genetic complementation experiments(Gardete et al., 2012). These data indicate that the decreasedvirulence in VISA is associated with the same mutated generesponsible for the reduced vancomycin susceptibility. VISAstrains with decreased or attenuated virulence fail to causeacute infections; however, decreased or attenuated virulence mayrepresent a “stealth” strategy to evade host immune surveillanceand promote clinical persistence (Gardete et al., 2012).

DIVERSE MUTATIONS IDENTIFIED IN VISABY WHOLE GENOME SEQUENCING

The molecular mechanisms for promotion vancomycinresistance in VISA are attractive because of the widespreadincidence of VISA globally. The best way to identify the geneticmutations in VISA is the whole genome sequencing of carefullyselected isogenic VSSA/VISA pairs or series from the clinicor derived from the laboratory. Table 1 shows the mutationsidentified by whole genome sequencing in hVISA/VISAstrains that may be responsible for the reduced vancomycinsusceptibility as compared with their progenitor VSSA. Betweenthe VISA strain JH9 and the VSSA strain JH1, the JH9 strainhad 35 point mutations in 31 genes (Mwangi et al., 2007). Inthe laboratory, a VISA could be obtained by step-wise selectionwith increasing concentrations of vancomycin in the culturemedia to exhibit a vancomycin MIC level of >32 µg/mL, such asVC40 [14], to the VRSA phenotype. Compared with its parentstrain RN42201mutS, VC40 had 79 mutations in 75 genes(Berscheid et al., 2014), thereby suggesting that the mutations inS. aureus genes may have cumulative effects that contribute to theVISA phenotype. Most studies usually reveal no more than 10mutations in isogenic VSSA/VISA pairs (Table 1). However, onepair revealed that only one mutation (WalR-K208R) is enoughto convert a VSSA to VISA (Howden et al., 2011). From thesame parent VSSA strain JH1, two in vitro selected VISA isolatesJH1R1 and JH1R2 have different mutations when compared witheach other, which also differed from the in vivo VISA isolate JH2(Vidaillac et al., 2013). These studies demonstrated that multiplepathways are involved in the generation of VISA from VSSA.

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Hu et al. Molecular Events in VISA

TABLE 1 | Mutations identified in hVISA/VISA strains comparied with the progenitor VSSA by whole genome sequencing.

VSSA/hVISA/VISA pairs

or series

Vancomycin

resistant

level (µg/mL)

Number of mutated

genes

Potential important mutations linked to VISA Year References

JH1/JH2/JH5/JH6/JH9 1/4/6/8/8 7/8/16/31 VraT-H164R, RpoB-D471Y+A473S+A477S+E478D

RpoC-E854K, PrsA-1C

2007 Mwangi et al., 2007

Mu3/Mu50 2/8 17 GraR-N197S, RpoB-H481Y, Fdh2-A297V, Sle1-167aa 2008 Neoh et al., 2008

Mu50�/Mu50 0.5–1/6–7 10 GraR-N197S, VraS-2341C 2009 Cui et al., 2009

N3151IP/H14 1/2 1 VraS-S329L 2009 Katayama et al., 2009

ISP794/AR376 2/4 3 Stp1-Q121C, VraS-G45R, YjbH-K231C 2011 Renzoni et al., 2011

N315LR5P1/LR5P1-V3 1.5/4 2 WalK-1Q371, ClpP-1N 2011 Shoji et al., 2011

JKD6000/JKD6001 1/4 7 WalR-A96T 2011 Howden et al., 2011

JKD6004/JKD6005 2/4 1 WalR-K208R 2011 Howden et al., 2011

JKD6009/JKD6008 2/4 10 WalK-G223D, GraS-T136I 2011 Howden et al., 2011

JKD6021/JKD6023 1/4 8 WalK-V268F 2011 Howden et al., 2011

JKD6052/JKD6051 1/4 7 RpoB-H481Y, SarR-A68T 2011 Howden et al., 2011

SG-S/SG-R 1.5/3 5 VraT-Y220C, YycH-A165D, VraG-G551E 2012 Gardete et al., 2012

A5937/A5940 1.5/4 6 Stp1-1131C, H481Y 2012 Cameron et al., 2012

A6264/A6226 2/3 13 DltA-S38R, ArlS-381C 2012 Cameron et al., 2012

A6300/A6298 2/4 8 Drp35-N83S 2012 Cameron et al., 2012

A9635/A9636/A9637/

A9638/A9639

1/1/2/3/4 1/2/5/4 VraT-N74D, VraG-A580V 2012 Cameron et al., 2012

A8117/A8118/A8392 1/4/8 3/5 WalK-R263C-S273N, WalK-1Q371, TcaR-I69S-K95N,

RpoB-S1052L

2012 Cameron et al., 2012

VSSA-A1/VISA-A2 1/8 6 Stp1-E18D19-duplication 2012 Passalacqua et al., 2012

JH1/JH1R1 1/3 4 WalK-G223D 2013 Vidaillac et al., 2013

JH1/JH1R2 1/3 1 RpoB-R484C+N641K 2013 Vidaillac et al., 2013

Mu3/Mu3 derived 45 VISA

isolates

3/6–12 1–4 mutations affecting

a total of 48 genes

BPB4-S140N, TarO-P94L, Cmk-A20G, etc. 2013 Matsuo et al., 2014

D32/D52/D56/D83/D90/D109 Not shown 1/1/4/6/4 WalK-Q369R, WalK-M220I, VraG-1L294N295 2014 Van Hal et al., 2014

109/2482 1.5/3 11 RpoB-A477V+S529L, AgrC-L1931C 2014 Chen et al., 2014

RN42201mutS/VC40 1.5/64 75 VraS-L114S+D242G, WalK-I544M, MprF-H224Y,

RpoD-D201N

2014 Berscheid et al., 2014

MW-2/SV-1 2/16 or 1/16 5 WalK-G223D, TarO-frameshift 2015 Hu et al., 2015

CH1/CH2/CH3/CH4/CH5/

CH6/CH7

2/2/2/3/3/3/4 0/1/3/2/2/2 YycH-1C, MprF-S295L 2015 Chen et al., 2015

13136p−m+/13136p−m+

V5/13136p−m+V20

1/8/16 8/9 Stp1-A143G, TarO-L670F 2015 Hattangady et al., 2015

8 MR/VR pairs 1–2/8–32 50–172 in different

pairs

Diverse mutations in GraS, RpoB, RpoC, WalK and etc. 2015 Ishii et al., 2015

∆N, N-terminal deletion; ∆C, C-terminal deletion.

However, the diverse genetic mutations that contribute to thealtered cell wall structures and increased vancomycin resistancein VISA need to be experimentally investigated.

EXPERIMENTALLY VERIFIED GENETICMUTATIONS IN CERTAIN GENESRESPONSIBLE FOR VISA EVOLUTION

Although hundreds of single nucleotide polymorphisms (SNPs)were discovered in VISA as compared with VSSA after thewhole genome sequencing of VSSA/VISA pairs or series, theexperimentally verified genetic mutations in certain genesresponsible for VISA evolution are still limited. Two methods

are usually used to functionally verify the role of a certainmutation for the promotion of vancomycin resistance in VISA.First, the mono- or bi-directional allelic exchange experimentreplaces the normal allele in VSSA with the mutated allele fromVISA or replaces the mutated sites in VISA with the normalallele from VSSA; subsequently, the results are used to evaluatewhether the allele swapping is responsible for vancomycinresistance. Some studies also conduct whole genome sequencingof the allele-swapping strains to exclude other mutations duringexperiments (Howden et al., 2011). The pKOR1 plasmid isusually used in these allele-swapping experiments because ithas a counter-selection marker and can be used to generatemultiple allele swapping events (Bae and Schneewind, 2006).For VISA clinical isolates that cannot be transformed, the

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Hu et al. Molecular Events in VISA

mutations are reconstituted in well-defined laboratory strains,such as N3151IP (Katayama et al., 2016). The constructedstrains are subject to vancomycin MIC determination, cell wallthickness measurement with electron microscopy, and othertests to verify and explore the mechanisms of gene mutationin vancomycin resistance (Cameron et al., 2012). Anothergenetic method is plasmid complementation with wild type ormutated genes in VSSA or VISA isolates; however, the copynumber of plasmids cannot be precisely controlled, and theresults of complementation are usually not consistent with theallele swapping results (Neoh et al., 2008; Matsuo et al., 2011).Regardless, plasmid complementation is easy and labor-saving;thus, this approach is still widely used for the verification ofloss-of-function mutations (Gardete et al., 2012; Matsuo et al.,2014).

Allelic exchange techniques or complementation methodshave experimentally verified the four categories of geneticmutations in VISA as summarized in Table 2. The first categoryincludes the genetic mutations in cell wall synthesis, hydrolysis,or remodeling genes, including sle1 and msrR (Katayamaet al., 2016). The second group includes the genetic mutationsin metabolic genes, including cmk and fdh2 (Matsuo et al.,2014; Katayama et al., 2016). The third category contains thegenetic mutations in transcription regulatory genes, includingyvqF/vraT-vraSR, graSR, walKR, and rpoB (Howden et al., 2014).The fourth group has the genetic mutations in post-translationalmodification genes, including stp1 and clpP (Shoji et al., 2011;Cameron et al., 2012).

Mutated Genes for Cell Wall Synthesis,Hydrolysis, and Teichoic Acid SynthesisAccelerated cell wall synthesis and decreased autolysisare alternative pathways for a thickened cell wall. Sle1 isthe hydrolase of N-acetylmuramyl-L-alanine amidase forpeptidoglycan biosynthesis and is involved in the cell wallseparation of S. aureus (Kajimura et al., 2005). Six mutationswere found in the VISA strain Mu50, but not in the VSSA strainN3151IP; one of these mutations is sle1 (167aa). The deleted67-aa polypeptide was localized to the LysM domain; thus, sle1(167aa) is a loss-of-function mutation. The introduction ofsle1(167aa) into N3151IP drastically decreased the autolyticactivity and converted the induced cell wall thickening toconstitutive cell wall thickening (Katayama et al., 2016). Loss-of-function mutations in cell wall hydrolysis genes, such as sle1, candirectly contribute to vancomycin resistance in VISA.

MsrR is a member of the LytR–CpsA–Psr (LCP) family,which attaches wall teichoic acid (WTA) to the peptidoglycanlayer (Chan et al., 2013). The deletion of msrR reducedWTA attachment and increased the cell size and aggregation(Hübscher et al., 2009). WTA controls staphylococcal autolysisby preventing the binding of autolysin to the assembled cellwall, but not to the septum (Schlag et al., 2010). Expression ofmsrR is increased by ≈four-fold upon vancomycin treatment(Gardete et al., 2006). Overexpression of msrR in the VSSAstrain N315 reduced vancomycin and teicoplanin susceptibility(Cui et al., 2005). The msrR (E164K) mutation is one of

six mutations identified in Mu50. The introduction of thismutation into N3151IP increased the vancomycin resistance(Katayama et al., 2016). The MsrR (E164K) mutation maypromote the attachment of WTA to the peptidoglycan layerand further prevent the binding of autolysin to the assembledcell wall, thereby decreasing cell wall autolysis and resistance tovancomycin. However, whether the MsrR (E164K) mutation is again-of-function mutation or a loss-of-function mutation is stillnot resolved.

WTA is a highly negative charged polymer; the downregulatedproduction of WTA may decrease the negative charge of thecell surface, thereby also contributing to vancomycin resistancevia electrical repulsion. The PBP4 is required for the highlycross-linked peptidoglycan synthesis. PBP4 is localized at thedivision septum; however, in the WTA synthesis gene tagOdeletionmutant, PBP4 was dispersed on the entire cell membraneand could not function properly; thus, the degree of cross-linking is decreased (Atilano et al., 2010). Furthermore, WTAalso controlled autolysin activity by influencing its localization(Schlag et al., 2010). The loss of all the WTA caused by tagOgene deletion enhanced autolysis (Schlag et al., 2010); however,the inhibition of the late WTA synthesis by Targocil decreasedautolysis and strongly induced cell wall stress stimulon genes,including pbpB and fmtA (Campbell et al., 2012). To obtaina more comprehensive view of hVISA-to-VISA conversion, 45high-level VISA isolates were selected by exposing Mu3 and itsrelated strains to 6 µg/mL vancomycin. Among the 45 VISAisolates, 32 have a single mutation in 20 genes, thereby indicatingthat these genes are directly involved in vancomycin resistance.Among the 32 single mutations, six mutations were identified inthe tarO, tarA, and tarL genes (Matsuo et al., 2014). A frameshift insertion mutation in tarO (llM) was also discovered inthe laboratory MW-2-derived VISA isolate SV-1 (Hu et al.,2015). These studies collectively indicate that mutations or theregulation of the WTA can contribute to vancomycin resistancein VISA.

The lipoteichoic acid (LTA) is another negatively chargedpolymer, which is linked to the cell membrane via a diglucosyl–diacylglycerol (Glc2-DAG) linkage. In the LTA synthesispathway, glucose 6-phosphate (G6P) is converted to α-G1Pby PgcA, then α-G1P is activated by GtaB to generate UDP-glucose; YpfP catalyzes the progressive addition of glucose todiacylglycerol (DAG) from UDP-glucose to yield the LTA anchorGlc2-DAG, which is flipped out by the LtaA protein. The lossof function of PgcA, GtaB, and YpfP did not inhibit LTAformation but altered the anchoring site and chain length of LTA(Gründling and Schneewind, 2007), thereby influencing the cellwall characteristics. In ∆ypfP, the autolysis activity is decreasedcompared with the wild type (Fedtke et al., 2007). Interestingly,one Mu3-derived VISA isolate has a C-terminal deletion ofGtaB, which lacks the catalytic core of GtaB. This loss-of-function mutation in GtaB blocks the G6P to α-G1P conversion;subsequently, G6P might be redistributed to the peptidoglycanprecursor synthesis. The bacterial two-hybrid approach revealedthat YpfP and LtaA can interact with numerous proteins involvedin cell division, peptidoglycan synthesis, or cell wall modification,including FtsA, FtsW, PBP2, PBP4, and DltD (Reichmann et al.,

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TABLE 2 | Experimental verified mutations in VISA.

Target Year Mutation Sites Parental strain Vancomycin MIC

(µg/mL) changes

Methods References

VraTSR 2009 VraS-S329L N3151IP 1→2 pKOR1 mediated allele swapping Katayama et al., 2009

2012 VraT-Y220C SG-R 3→1.5 pGC2 mediated complementation

with wild type VraT

Gardete et al., 2012

2012 VraS-2341 SG-rev 1→3 pGC2 mediated complementation

with wild type VraS

Gardete et al., 2012

2014 VraS-L114S+D242G NCTC8325 1.5→4 Temperature-sensitive shuttle

vector pMAD mediated allele

swapping

Berscheid et al., 2014

GraSR 2008 GraS-T136I JKD6009 2→6 pKOR1 mediated allele swapping Howden et al., 2008

2008 GraR-N197S Mu3 2→4 pYT3 mediated overexpression Neoh et al., 2008

2011 GraS-T136I JKD6009 1.5→2 pKOR1 mediated allele swapping Howden et al., 2011

WalKR 2011 WalK-G223D JKD6009 1.5→3 pKOR1 mediated allele swapping Howden et al., 2011

2011 WalR-K208R JKD6004 or JKD6005 1.5→4 or 4→1.5 pKOR1 mediated allele swapping Howden et al., 2011

2011 WalK-1Q371 LR5P1 1.5→3 pKOR1 mediated allele swapping Shoji et al., 2011

2015 WalK-G223D MW2 2→4 pBTs mediated allelic replacement,

pBTs is derived from pBT2 and

pKOR1

Hu et al., 2015

ClpP 2011 ClpP-1N LR5P1 1.5→2 pKOR1 mediated allele swapping Shoji et al., 2011

Stp1 2012 Stp1 deletion A5937 1.5→3 pKOR1 mediated gene deletion Cameron et al., 2012

2012 Stp1-E18D19 duplication Strain A2 6–8→3 pOS1-Plgt mediated wild type

Stp1 complementation

Passalacqua et al., 2012

Cmk 2014 Cmk-A20G, CmK-T(-13)A Mu3 2→8, 3→8 pKOR1 mediated allele swapping Matsuo et al., 2014

2014 Cmk-A20G, CmK-T(-13)A Mu3p27V6–10 8→2, 8→3 Introduce the pND50-cmk plasmid

into the VISA isolates

Matsuo et al., 2014

VraS+GraR 2009 VraS-I5N+GraR-N197S Mu50� 4→6 pKOR1-mediated allele swapping Cui et al., 2009

GraS+WalK 2011 GraS-T136I+ WalK-G223D JKD6009 1.5→4 pKOR1-mediated allele swapping Howden et al., 2011

GraR+RpoB 2011 GraR-N197S+RpoB-

H481Y

Mu3 2→6 pKOR1-mediated allele swapping Matsuo et al., 2011

WalK+ClpP 2011 WalK-1Q371+ ClpP-1N LR5P1 1.5→4 pKOR1 mediated allele swapping Shoji et al., 2011

VraS+Stp1+YjbH 2011 VraS-G45R+Stp1-

Q121C+YjbH-K231C

ISP794 2→4 Plasmid mediated gene

replacement and bacteriophage

transduction mediated triple

mutant construction

Renzoni et al., 2011

VraS+GraR+

RpoB+Fdh2+

Sle1+MsrR

2016 VraS-S329L+GraR-N197S

+RpoB-H481Y+Fdh2-

A297V+Sle1-167aa+

MsrR-E164K

N3151IP 1→12 pKOR1 mediated allele swapping Katayama et al., 2016

The vancomycin MIC levels determination methods are not consistent with each other in different studies.

2014). Overall, mutations in the synthesis of peptidoglycan,WTA, and LTA or the remodeling processes can directly orindirectly contribute to vancomycin resistance in VISA.

Mutated Genes Involved in StaphylococcalMetabolismCell wall synthesis consumes high amounts of substrate andenergy. To build a thickened cell wall, the demand for cell wallbiosynthetic precursors is increased in VISA, which requires theadjustment of cellular metabolism. VISA strains have impairedacetate catabolism (Nelson et al., 2007). Genetically distinctVISA strains are also associated with specific and reversiblemetabolic alterations (Alexander et al., 2014). The abundanceof six metabolites from the urea cycle, the pentose phosphatepathway, and the TCA cycle was altered in the JH series and the

SG series of VISA isolates (Alexander et al., 2014). These alteredmetabolites are directly or indirectly linked to the biosynthesisof cell wall precursors. Mutations in metabolic genes can directlyadjust cellular metabolism to support the cell wall synthesis.

The cmk gene product functions in the pyrimidine synthesispathway. A decrease in Cmk activity is expected to increaseUTP, which is required for the synthesis of a key peptidoglycanintermediate UDP-GlcNAc. Two mutated forms of the cmk genehave been introduced to Mu3, thereby converting the hVISAisolate Mu3 to VISA isolates. One mutant is the Cmk-A20Gmutation; the other is a T-to-A mutation in the Shine-Dalgarno(SD) sequence of Cmk (Matsuo et al., 2014). The Cmk-A20Gmutation decreased the Cmk activity, whereas a mutation in theSD sequence decreased the translation of Cmk. A decrease in theCmk activity or Cmk protein levels probably caused increased

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vancomycin resistance by increasing the supply of cell wallbiosynthesis intermediate UDP-GlcNAc (Matsuo et al., 2014).Cmk catalyzed the formation of cytidine diphosphate (CDP).CDP is required for DNA/RNA synthesis and the formationof the WTA synthesis precursor CDP-glycerol. Peptidoglycanand WTA production depend on the lipid carrier undecaprenylphosphate; the Cmkmutation might suppress theWTA synthesispathway and enhance peptidoglycan synthesis.

Fdh2 is a putative formate dehydrogenase. The introductionof an Fdh2-A297V mutation into N3151IP with theaforementioned Sle1(167aa) converted the inducible cellwall thickening to constitutive cell wall thickening (Katayamaet al., 2016). Therefore, mutations in metabolic genes can directlyinfluence cell wall synthesis and contribute to vancomycinresistance in VISA. Several other mutated metabolic proteinshave been identified in VISA isolates, including proteins involvedin translation, nucleotide metabolism, amino acid biosynthesis,and so on. These proteins may also directly adjust the cellmetabolism to support cell wall synthesis in VISA.

Mutated Genes for TranscriptionalRegulationAmong the cell wall synthesis or hydrolysis genes, the metabolicgenes are dynamically regulated by transcriptional factors andother regulators. Hundreds of genes (≈10% of the wholeS. aureus genome) from several functional categories aredifferentially expressed in VISA and VSSA. Previous studieshave directly identified mutations in transcriptional regulatorsthat may be the key players of vancomycin resistance in VISAisolates. Most mutations in vraTSR, graSR, walKR, and rpoB havebeen experimentally verified to be responsible for promotingvancomycin resistance in VISA.

Mutation Activated VraTSR SystemVraSR is characterized as vancomycin resistance-associatedbecause the VraSR operon is upregulated in the first reportedVISA isolate Mu50 (Kuroda et al., 2000). The operonencompassing VraSR consists of four genes. The orf1 gene isnot required for cell wall stress stimulon, whereas VraT/YvqF–VraS–VraR is a three-component system that regulates cell wallstress stimulon (McCallum et al., 2011; Boyle-Vavra et al., 2013).VraTSR positively modulates the cell wall biosynthesis pathwayin S. aureus (Kuroda et al., 2003). After induction by a cellwall synthesis inhibitor, VraR activates the vra operon and 46other genes for the cell wall stress regulon; some of these genes,such as pbpB and fmtA, encode known or putative cell wallsynthesis proteins (Gardete et al., 2006). The use of an IPTG-inducible promoter controlled pbpB revealed that the VraTSRsystem might directly sense a cell wall synthesis step catalyzed byPBP2 (Gardete et al., 2006). The consensus binding site of VraRhas been deduced (5′-ACT-N3-AGT-3′ or 5′-TGA-N3-TCA-3′)(Belcheva et al., 2009). Chromatin immunoprecipitation showedthat the promoters of pbpB, murZ, and sgtB could directly bebinded by the VraR proteins, whereas direct binding was notpossible with fmtA (Sengupta et al., 2012). Upregulation of VraSRand the VraSR-dependent cell wall stimulon is linked to theincreased vancomycin resistance phenotypes of several clinical

VISA isolates (Kuroda et al., 2000; McAleese et al., 2006; Katoet al., 2008, 2010). By contrast, deletion of the vraSR operonenhanced the sensitivity to vancomycin and other cell walltargeting antibiotics (McCallum et al., 2011; Boyle-Vavra et al.,2013).

The VraT-Y220C, VraS-I5N, VraS-S329L, and VraS-L114S-D242G mutations have been experimentally associated with theVISA phenotype. Whole genome sequencing of the susceptibleSG-S, the resistant strain SG-R, and the spontaneous revertantstrain SG-rev in an isogenic VISA-type series of ST8-USA300isolates revealed that the VraT-Y220C mutation mediatesthe vancomycin-resistant phenotype, whereas a prematuretermination of the VraS at amino acid 234 reverts thevancomycin-resistant phenotype (Gardete et al., 2012). VraS has347 amino acids but the histidine kinase domain is lost whenshortened to 234 amino acids. These data suggest that VraT isa negative regulator of VraSR, and VraT-Y220C is a loss-of-function mutation. Complementation of the VISA isolate withwild-type vraT can revert the vancomycin-resistant phenotype,whereas complementation of the reverted VSSA isolate withwild-type VraS can promote the vancomycin-resistant phenotype(Gardete et al., 2012).

The introduction of the VraS-I5N or VraS-S329L mutationinto a VSSA isolate increased vancomycin resistance (Cui et al.,2009; Katayama et al., 2016). The reconstitution of the VraS-L114S-D242G allele into the VSSA strain NCTC8325 increasedresistance to vancomycin (Berscheid et al., 2014). Furthermore,the cell wall stress regulon controlled by VraSR is upregulated inthese VISA isolates. All these studies indicate that mutations inthe VraTSR system cause its activation and the upregulation ofthe cell wall stress regulon, thereby increasing cell wall synthesisand vancomycin resistance in VISA.

Mutation Activated GraSR SystemThe GraSR system is a two component system (TCS) associatedwith glycopeptide resistance (Cui et al., 2005). The GraSRproteins interact with GraX and the VraFG ABC transporter toform a five-component system for cationic antimicrobial peptide(CAMP) resistance (Falord et al., 2012a). The GraSR systemis required for CAMP resistance, such as resistance to humandefensins. Resistance to CAMPs is mediated by the D-alanylationof cell wall teichoic acids by the DltABCD enzymes and theMprF-dependent lysylination of phoshpatidylglycerol (Ernst andPeschel, 2011). GraR can self-dimerize; upon phosphorylationby GraS on the D51 residue, GraR binds to the promotersof vraFG, dltABCD, and mprF to initiate their transcription.A highly-conserved 10 base pair palindromic sequence (5′-ACAAATTTGT-3′) is the putative GraR-binding site, which islocated in the upstream sequences ofmprF, dltABCD, and vraFGgenes.

The GraSR system is upregulated in VISA isolates (Cui et al.,2005). The deletion of GraSR or VraFG caused hypersensitivity tovancomycin, increased autolysis, and produced a more negativenet surface (Meehl et al., 2007). The Mu50 strain has a GraR-N197S substitution; the introduction of this substitution onthe plasmid pYT3 vector can convert the hVISA strain Mu3to achieve high vancomycin resistance similar to that of the

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VISA strain Mu50 (Neoh et al., 2008). However, the GraR-N197S substitution on the pYT3 vector is a high-copy version;the introduction of a single copy of GraR-N197S into Mu3did not increase vancomycin resistance to the level of VISA(Matsuo et al., 2011). Thus, the GraR N197S only has a marginaleffect on vancomycin resistance. The GraR-N197S substitutionand the RpoB-H481Y mutation can convert Mu3 to the fullvancomycin resistance level of Mu50 (Matsuo et al., 2011).The GraS-T136I mutation can also increase the vancomycinresistance levels (Howden et al., 2008). However, GraS-T136Ialone is not sufficient to convert VSSA to the full vancomycinresistance of VISA. The combination of GraS-T136I and WalK-G223D is required to achieve the full vancomycin resistance levelof VISA (Howden et al., 2011). All these studies indicate that theactivated GraSR system can increase vancomycin resistance byupregulating the dltABCD andmprF genes.

Mutation Downregulating the WalKR System

Expression or Activity Contributed to Vancomycin

Resistance in VISAAs the only essential TCS for the viability of S. aureus, theWalKRsystem connects cell wall biosynthesis with cell division. WalKhas two transmembrane helices and a C-terminal kinase domain.The N-terminal Per–Arnt–Sim (PAS) domain may be involvedin signal sensing. WalR controls the expression of its regulon byforming a head-to-head dimer of the receive domains, which ispaired with a head-to-tail dimer of the winged helix-turn-helixmotifs that bind to the tandem DNA repeats of the binding site(Dubrac et al., 2008). The potential consensus DNA recognitionsequences of WalR consist of two hexanucleotide direct repeats(5′-TGT(A/T)A(A/T/C)-N5-TGT(A/T)A (A/T/C)-3′) (Dubracet al., 2008). In VISA isolates, the expression levels of altA,sle1, lytM, and several genes encoding CHAP domain-containingproteins were down-regulated. The genes for AtlA, Sle1, LytM,and the CHAP domain-containing proteins are repressed byTCSs, such as LytSR and ArlSR, and positively regulated bythe WalKR. Promoter analysis demonstrated that atlA, sle1, andlytM have the consensus sequence for WalR binding. Expressionanalysis by WalKR starvation or the constitutive form of WalRsupported that atlA, sle1, and lytM are directly transcribed by theWalKR system in S. aureus (Dubrac et al., 2007; Delauné et al.,2012).

Two studies examined an IS256 insertion into the walKRpromoter and revealed that the upregulation ofwalKR expressionwill decrease resistance levels to vancomycin, whereas thedownregulation of walKR expression will increase the resistancelevels of vancomycin (Jansen et al., 2007; McEvoy et al., 2013).Jansen et al. (2007) compared two VISA isolates SA137/93A(MIC= 8µg/mL) and SA137/93G (MIC= 12µg/mL) and foundthat SA137/93A has an IS256 insertion in the predicted promoterregion. However, this IS256 insertion generates a potentiallystronger promoter, which upregulated the walKR expressionof SA137/93A; thus, the upregulation of WalKR produceslow levels of vancomycin resistance (8 vs. 12µg/mL), therebyindicating that walKR is a negative regulator of vancomycinresistance (Jansen et al., 2007). An IS256 insertion to the 5′-untranslated region (5′-UTR) of walKR reduced the expression

of walKR by ≈50%; the downregulation of walKR will increasethe vancomycin resistance level of VSSA isolates (McEvoy et al.,2013). The removal of IS256 could revert the VISA phenotype toVSSA, thereby confirming that the reduced expression of WalKRinduced the VISA phenotype. These studies collectively suggestthat the downregulation of walKR expression contributes to thedevelopment of vancomycin resistance in VISA.

Several studies examined the mutated forms of WalKR invancomycin resistance of VISA. In a laboratory-derived VISAstrain, Shoji et al. (2011) found that the walK∆Q371 alleleinfluences vancomycin resistance, but this allele is not sufficientfor full vancomycin resistance. However, the walKR mutationwas the most frequent in VISA strains. Genome sequencing offive VSSA/VISA pairs revealed that the VISA in four of these pairscarried mutations in the WalKR system (Howden et al., 2011).Subsequently, allelic replacement demonstrated that WalK-G223D with GraS-T136I could convert VSSA to full intermediatevancomycin resistance similar to VISA. The allelic replacementof WalR-K208R can also convert VSSA to full intermediateresistance to vancomycin (Howden et al., 2011). Howden et al.(2011) proposed that mutations in WalKR may downregulateits activity. SV-1 is a laboratory-derived VISA strain from CA-MRSA MW2 with the same WalK-G223D mutation as theclinically-isolated strain JKD6008 (Hu et al., 2015). The WalK-G223D mutation had decreased autophosphorylaton of WalK,thereby reducing the phosphorylation of the WalR responseregulator and decreasing the binding activity of WalR to thealtA promoter based on the electrophoretic mobility shift assay(Hu et al., 2015). Therefore, the downregulated expression orimpaired activity of the WalKR system can mediate vancomycinresistance in VISA by downregulating the expression of autolysingenes.

Mutation Altered the Activity of RpoBThe rpoB gene encodes the β subunit of RNA polymerase, whichis active in catalysis. Mutations in RpoB might influence thetranscriptional activity of the whole RNA polymerase. The mostextensively studied mutation of RpoB in VISA is the H481Ysubstitution. The introduction of a single copy of the GraRN197S into the Mu3 is not enough to achieve the vancomycinresistance level of the VISA isolate Mu50 (Matsuo et al., 2011).Targeted sequencing of the rifampicin resistance-determiningregion (RRDR) showed that Mu50 has an H481Y mutation atRpoB. Genetic swapping studies confirmed that the RpoB-H481Ymutation is responsible for the emergence of the VISA phenotype(Matsuo et al., 2011). The RpoB-H481Y mutation causes globaltranscriptional changes, which promote antimicrobial peptideresistance, with attenuated virulence in a murine bacterimiamodel (Gao et al., 2013). Mutations in other subunits of RNApolymerase such as RpoC or RpoD, have also been foundin VISA isolates (Matsuo et al., 2014). The ST239-MRSA-III-t030 clone is prevalent in China; most ST239-MRSA-III-t030strains harbor double substitutions in RpoB (H481N+L466S)that confer resistance to rifampicin, but not to vancomycin(Zhou et al., 2012; Shang et al., 2016). Therefore, the RpoB-H481N mutation may not confer vancomycin resistance similarto that of the RpoB-H481Y mutation; otherwise, a second

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mutation (L466S) could compensate for the effect of H481Non vancomycin resistance. This hypothesis is worthy of furtherinvestigation.

SarA/MgrA Family Proteins in VISAThe staphylococcal accessory protein A (SarA) family of globalregulatory proteins control virulence, antibiotic resistance, cellwall synthesis, and other defensive pathways. SarA and MgrAare negative regulators of murein hydrolases (Ingavale et al.,2003). SarV is repressed by SarA and MgrA and is involvedin autolysis (Manna et al., 2004). The sarA deletion mutantdisplayed enhanced resistance to vancomycin (Sun et al., 2012).Moreover,mgrA overexpression increased vancomycin resistancein S. aureus (Cui et al., 2005). An A68T mutation in SarR wasidentified in a VISA clinical isolate; however, this mutation hasnot been experimentally verified (Howden et al., 2011).

In the constitutive WalR mutant, the expression of sarS, sarT,and icaR was repressed by the WalKR system (Delauné et al.,2012). The sarS and sarR expression is also positively controlledby GraSR (Falord et al., 2012b). The msaABCR operon canpositively regulate the expression of sarA to promote biofilmformation and enhance virulence while negatively regulatingautolysis in S. aureus (Sambanthamoorthy et al., 2006). Theinactivation of the msaABCR operon in three different VISAisolates increased the susceptibility to vancomycin and reducedthe vancomycin-binding capacity (Samanta and Elasri, 2014).These studies collectively indicate that the SarA family proteinsare crucial regulators in VISA.

CcpA and CcpE, Potential Regulators in VISAThe catabolite control protein A (CcpA) is the major regulator ofcarbon catabolite repression (CCR). In the presence of glucose orother preferred carbon sources, CcpA forms a complex with S46-phosphorylated Hpr protein. The CcpA–Hpr-S46(P) complexbinds to the catabolite responsive element (cre) sequencesof diverse genes to activate or repress their expression. Theexpression of ccpA is negatively self-regulated (Leiba et al., 2012).The expression of several genes is affected by ccpA inactivationin the absence of glucose, thereby indicating that the functionof CcpA is not restricted to CCR and includes virulence andantibiotic resistance regulation (Seidl et al., 2009). CcpA canalso regulate numerous virulence factors (Seidl et al., 2006).The expression of the urease operon is downregulated in ∆ccpAcompared with the wild type, which is consistent with results ofthe urease activity assay (Seidl et al., 2009).

Transcription analysis revealed that the phosphorylationtransfer system (PTS) of different carbon sources is usuallyinfluenced by antibiotic treatment or the vraSR, graSR, andwalKR systems. The expression of PTS genes is directly controlledby the CcpA protein. CcpA represses the tricarboxylic acid (TCA)cycle, which is required for the full oxidation of glucose; CcpAalso regulates the genes involved in the glycolytic pathway andmetabolism (Seidl et al., 2009). Although the CcpA mutationwas not observed in VISA isolates, the deletion of ccpA reducedteicoplanin resistance levels in a glycopeptide-intermediate S.aureus strain (Seidl et al., 2006). The altered central metabolism

in VISA can also regulate CcpA activity, which can be fine-tuned by small molecule effectors, such as G6P and fructose1,6-bisphosphate (FBP), which both enhance the DNA-bindingactivity of the CcpA–Hpr-S46(P) complex (Schumacher et al.,2007). However, the G6P and FBP levels significantly decreasedin the ∆stp1 strain, which has increased vancomycin resistance(Liebeke et al., 2010). Mutations in metabolic genes or thealtered central metabolism in VISA isolates might control geneexpression via the CcpA protein.

Catabolite control protein E (CcpE) can also affects thecentral metabolism as well as virulence. CcpE is the first positiveregulator of TCA cycle reported in S. aureus by activating theexpression of acotinase gene (citB) (Hartmann et al., 2013). Inaddition, CcpE positively regulate the expression of acetyl-CoAcarboxylase (AAC) gene, while AAC catalyzes the committed stepin fatty acid biosynthesis (Ding et al., 2014). On the other hand,CcpE negatively control the expression of many virulence genes(Ding et al., 2014). Intriguingly, CcpE is allosterically activatedby the TCA cycle intermediate citrate (Ding et al., 2014). Thealtered central metabolism observed in VISA might control geneexpression via the CcpE protein and vice versa, though no geneticmutations occured in CcpE have been observed in VISA isolates.

Mutated Genes for ProteinPhosphorylation or Degradation MediateVancomycin Resistance in VISAAside from transcriptional control, the protein phosphorylationand degradation processes are widely used as regulatorymechanisms. The reversible phosphorylation anddephosphorylation of proteins by Stk1/Stp1 have importantroles in diverse cellular processes of S. aureus (Ohlsen andDonat, 2010). Moreover, the ATP-dependent ClpP proteaseinvolved in intracellular proteolysis plays important roles instress response, metabolism and cell wall synthesis (Frees et al.,2014). Mutations in stp1 or clpP have been associated withvancomycin resistance in VISA.

Mutations in stk1/stp1The Stk1 (PknB)/Stp1 (PP2C) pair is a eukaryotic-like Ser/Thrkinase/phosphatase system in bacteria. Stk1 and Stp1 areusually found to function together to regulate the reversiblephosphorylation of substrates. Stk1 is also a substrate of Stp1(Débarbouillé et al., 2009). In S. aureus, Stk1 is a membrane-bound protein, whereas Stp1 is a cytosolic protein. The N-terminal domain of Stk1 inside the cytoplasm is associatedwith kinase activity, whereas the C-terminal domain of Stk1has three penicillin-binding protein and serine/threonine kinase-associated (PASTA) domains, which are hypothesized to respondto cell wall signals. Biophysical and structural studies revealedthe direct interactions of the PASTA domain of Stk1 withmuropeptides (Ruggiero et al., 2012). The deletion of stk1,stp1, or both can influence cell wall structure and antibioticsusceptibility. The stp1 deletion mutant displayed a thickenedcell wall and increased resistance to lysostaphin (Beltramini et al.,2009). Furthermore, the stk1 deletion mutant was more virulentthan the parental strain in a murine cutaneous infection model

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(Tamber et al., 2010). By contrast, the stp1 deletion mutant wasavirulent in a mouse sepsis model (Cameron et al., 2012).

Three studies examined the role of protein phosphatase Stp1in VISA isolates or in isolates with decreased susceptibilityto teicoplanin (Renzoni et al., 2011; Cameron et al., 2012;Passalacqua et al., 2012). In the three studies, the loss of stp1function via the deletion of stp1 or a premature stop codonincreased the vancomycin and teicoplanin resistance levels. Atwo amino-acid insertion mutation at the highly conservedmetal-binding domain of Stp1 was discovered by whole genomesequencing between the VISA isolate and its parental VSSAisolate from a single patient with endocarditis (Passalacqua et al.,2012). The two amino-acid insertion mutation may lead tothe inactivation of Stp1 activity. The complementation of theVISA isolate with a wild copy of Stp1 reduced the vancomycinMIC (Passalacqua et al., 2012). Comparative genomics of acollection of VSSA/VISA isogenic pairs revealed the loss-of-function mutations in the stp1 gene of VISA isolates. Theconstruction of an stp1 deletion mutant showed that Stp1 isinvolved in both vancomycin resistance and virulence (Cameronet al., 2012). A premature stop codon mutation and two othermutations were identified in a laboratory-derived teicoplanin-resistant S. aureus isolate. Complete genetic analysis revealed thatthe loss-of-function mutation in Stp1 contributed to teicoplaninand vancomycin resistance (Renzoni et al., 2011).

The transcriptome analysis of stk1 deletion strains revealedthat Stk1 can regulate many genes involved in nucleotidebiosynthesis, cell wall metabolism, and autolysis (Donat et al.,2009). Several studies demonstrated that Stk1/Stp1 can crosstalk with TCSs and other transcription factors (Table 3). VraRis a substrate of Stk1 (Ling et al., 2014); the phosphorylationof VraR at multiple sites (T106, T119, T175, and T178) byStk1 negatively regulates its dimerization formation and DNA-binding activity, which can downregulate the VraSR system.Stk1/Stp1 can also regulate protein cysteine-phosphorylation ofthe SarA/MgrA family (SarA, MgrA, and SarZ) global regulators,which are involved in virulence and antibiotic resistance (Sunet al., 2012). In the stp1 mutant, the phosphorylation stateof SarA is increased. However, the binding to promoters islost, thereby indicating that SarA phosphorylation attenuatesits transcriptional activity. Another study revealed that CcpAis a substrate of the Stk1/Stp1 pair; the phosphorylation ofCcpA (T18, T33) also decreased its DNA-binding activity (Leibaet al., 2012). By contrast, GraR can be phosphorylated byStk1 at T128, T130, and T149 in the DNA-binding domain,but phosphorylation at these sites increased the DNA-bindingactivity of GraR (Fridman et al., 2013). Consequently, theexpression of dltABCD operons is upregulated; these operonsencode proteins to catalyze the D-alanyl esterification of thepolyglycerol phosphate LTA and the polyribitol phosphate WTA(Fridman et al., 2013). The increased positive charge of LTAand WTA play important roles in exclusion of the cationicantibiotics such as vancomycin (Fridman et al., 2013). Thestage V sporulation protein G (SpoVG) homolog of S. aureusmodulates virulence factor synthesis and antibiotic resistance.The deletion of spoVG reduced teicoplanin and vancomycinresistance; although the level of resistance to teicoplanin was

more pronounced than that to vancomycin (Schulthess et al.,2009). SpoVG can directly bind to the putative promoters of lytN,femA, and lytSR, and modulates oxacillin resistance in MRSAstrain N315 (Liu et al., 2016). A recent study found that SpoVGis also subject to phosphorylation by Stk1; the Stk1-mediatedphosphorylation markedly enhanced the DNA-binding activityof SpoVG (Bischoff et al., 2016). Therefore, the loss of stp1will cause the irreversible phosphorylation of several importanttranscription factors involved in vancomycin or teicoplaninresistance and virulence.Whether CcpE is regulated by Stk1/Stp1awaits further investigation.

Metabolomic studies found that the levels of cell wall synthesisprecursors were significantly changed in the S. aureus stk1 orstp1 mutant (Liebeke et al., 2010). For instance, the levels offructose-1,6-bisP, glucose-6-P, and fructose-6-P are significantlydecreased in the stp1 deletion mutant as compared with the wildtype (Liebeke et al., 2010). This study indicated that metabolicenzymes might also be subjects for phosphorylation by Stk1. Stk1can directly phosphorylate adenylosuccinate synthase (PurA)and downregulate its enzymatic activity (Donat et al., 2009).Autoinducer-2 (AI-2) is an important signaling molecule inquorum-sensing system of S. aureus, its synthase (LuxS) canbe inactivated by Stk1 phosphorylation at T14 (Cluzel et al.,2010). The substrates of Stk1/Stp1 in S. aureus are summarizedin Table 3.

Mutations in Genes Encoding Proteolytic ProteinsClpP is a proteolytic subunit of the ATP-dependent Clp protease.A core proteolytic chamber is formed by ClpP and flanked byseveral ATPases, including ClpC, ClpX, ClpB, and ClpL. TheATPases determine substrate specificity and perform chaperonactivities. The ClpP and Clp ATPases are involved in stressresponse, virulence, metabolism, and antibiotic resistance (Freeset al., 2014).

A 144 bp deletion of clpP caused the loss-of-function of ClpPwith the WalK Q371 single amino acid deletion, which wasfound to mediate vancomycin resistance in a laboratory-derivedVISA isolate (Shoji et al., 2011). The deletion of ClpP affectsthe expression of several important regulatory genes, includingagr, sigB, sarT, and walKR (Shoji et al., 2011). Proteomics hasrevealed that numerous proteins are substrates of ClpC or ClpP(Feng et al., 2013; Graham et al., 2013), including proteinsinvolved in cell wall synthesis, hydrolysis, and transcriptionalregulation (Table 4). The cell wall synthesis proteins, such asPBP2, FemA, and FemB, and the autolysins, including Sle1and Atl, can be hydrolyzed by ClpP; however, accumulatedautolysins are catalytically inactive in the ClpP mutant, andthe underlying mechanisms remain elusive (Feng et al., 2013).Transcription factors, such as CcpA,HprK (co-repressor of CcpAkinase), and CodY, are also substrates of ClpP or ClpC in S.aureus (Frees et al., 2014). The effects of proteolytic proteins,such as ClpP, on vancomycin resistance in VISA may occur viatheir regulational roles on the transcription factors. By contrast,ClpC, ClpP, and ClpB can also be repressed by the GraSRsystem (Falord et al., 2012b). However, ClpB is activated bythe WalKR system, whereas ClpC is repressed (Delauné et al.,2012).

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Hu et al. Molecular Events in VISA

TABLE 3 | Summary of substrates of Stk1/Stp1 determined in S. aureus.

Substrates Function Phosphorylation site(s) Effects of phosphorylation References

VraR Vancomycin-resistance-associated response

regulator VraR

Thr106, Thr119, Thr175, Thr178 Decreased DNA-binding properties Canova et al., 2014

SarA, SarA/MgrA family transcriptional regulators Cys9 Decrased DNA binding activity Sun et al., 2012

MgrA, SarA/MgrA family transcriptional regulators Cys12 Decrased DNA binding activity Sun et al., 2012

SarZ SarA/MgrA family transcriptional regulators Cys13 Decrased DNA binding activity Sun et al., 2012

CcpA Catabolite control protein A Thr-18, Thr-33 Electrophoretic mobility shift assays

demonstrated that the CcpA DNA

binding activity was completely

abrogated for the phosphorylated CcpA

Leiba et al., 2012

GraR a two-component system involved in

resistance to cationic antimicrobial peptides

Thr128, Thr130, Thr149 Increased DNA binding activity Fridman et al., 2013

SpoVG Modulator of virulence factor synthesis and

antibiotic resistance

Thr4, Thr13, Thr24, Ser41 Enhanced the DNA binding activity Bischoff et al., 2016

LuxS Autoinducer-2 synthase Thr14 The enzymatic activity of the

phosphorylated isoform of LuxS was

abrogated compared to that of

non-phosphorylated LuxS

Cluzel et al., 2010

PurA Adenylosuccinate synthase involved in

synthesis of AMP

Not determined Decreased enzymatic activity of PurA Donat et al., 2009

TABLE 4 | Summary of ClpP or ClpC substrates related to vancomycin

resistance in VISA.

Protein Function

name

Sle1 Autolysin precursor

Atl Bifunctional autolysin precursor

Glck Glucokinase

GlmS Glucosamine–fructose-6-phosphate aminotransferase, isomerizing

GlmM Phosphoglucosamine mutase

MurC UDP-N-acetylmuramate–alanine ligase

MurG UDP-glucose diacylglycerol glucosyltransferase

MurI Glutamate racemase

MurE UDP-N-acetylmuramoylalanyl-d-glutamate–2,6-diaminopimelate ligase

FemA Formation of the pentaglycine cross bridge

FemB Formation of the pentaglycine cross bridge

Pbp2 Penicillin-binding protein 2, peptidoglycan cross linking

SigA RNA polymerase sigma factor

RpoA DNA-directed RNA polymerase, alpha subunit

RpoB DNA-directed RNA polymerase, beta subunit

CodY GTP-sensing transcriptional pleiotropic repressor

SaeR Response regulator SaeR

CcpA Catabolite control protein A

AgrA Staphylococcal accessory gene regulator A

RsbW Serine-protein kinase RsbW

Other Genes Involved in VancomycinResistanceThe airSR genes encode an important TCS involved in cellwall biosynthesis and vancomycin resistance. The deletion ofairSR showed reduced viability of S. aureus in the presence ofvancomycin (Sun et al., 2013). The teicoplanin resistance factor A(TrfA) also affects vancomycin and oxacillin resistance (Renzoni

et al., 2009). TrfA is a homolog of the MecA adaptor proteinof the ClpC ATPase and is involved in the proteolysis. Thetranscription control of trfA is mediated by the thiol/oxidativestress global regulator Spx (Jousselin et al., 2013). The proteolyticdegradation of Spx is governed by the YjbH protein, which isan adaptor protein of the ClpXP protease that was mutated in alaboratory-derived VISA isolate (Renzoni et al., 2011). Thus, theYbjH–Spx–TrfA cascade is important for vancomycin resistancein VISA isolates. The tcaA gene encodes a transmembraneprotein, which is in the same operon as tcaB and tcaR. TcaBis a multidrug efflux pump, whereas TcaR is a transcriptionalregulator of virulent determinants. The inducible expression oftcaA is dependent on VraSR (Chen et al., 2016). The inactivationof tcaA can increase the vancomycin resistance of S. aureusisolates (Maki et al., 2004).

CONCLUSIONS AND A PROPOSEDMODEL

Given the increasing use of vancomycin, VISA emerged in themid or late 1980s and has become a global health threat. Althoughthe mechanisms for the promotion of vancomycin resistance inVISA are diverse, VISA exhibit several common characteristics,such as thickened cell walls, reduced autolysis, and attenuatedvirulence. The diverse mutations in certain genes identified inVISA imply the multiple complicated evolutionary pathwaysfor VISA. We proposed a multiple-hit model for vancomycinresistance in VISA (Figure 3). On one hand, the mutated andconstitutively activated VraTSR and GraSR systems increased thetranscription of genes for cell wall synthesis and modification,including pbpB, dltABCD, and mprF, thereby increasing thecell wall biosynthesis and the D-alanyl esterification of LTAand WTA. The mutated and actively downregulated WalKRsystem decreased the expression of autolysins and PBP4, thereby

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Hu et al. Molecular Events in VISA

FIGURE 3 | Molecular events in VISA. Key regulatory proteins and cell wall synthesis processes and their enzymes related to vancomycin resistance in VISA. WTA,

wall teichoic acid; LTA, lipoteichoic acid; Glc, glucose; GlcN-6-P, glucosamine-6-phosphate; GlcNAc, N-acetylglucosamine; MurNAc, N-Acetylmuramic acid; DAG,

diacylglycerol.

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Hu et al. Molecular Events in VISA

decreasing autolysis and the muropeptide cross-linking in VISA.The mutation and loss of function in Stp1 led to irreversiblephosphorylation of several transcriptional factors by Stk1, suchas GraR, VraR, CcpA, and SarA, which will directly or indirectlyinfluence vancomycin resistance in VISA. The mutation and lossof function in ClpP upregulated the transcription factors andcell wall synthesis proteins, such as PBP2, FemA, and FemB.Mutations occurred in the cell wall synthesis or hydrolysisgenes, such as msrR and sle1, which could directly influencecell wall synthesis; mutations in the metabolic genes, such asfdh2, could adjust the cell metabolism to support cell wallsynthesis precursors. These mutated genes collectively influencedthe balance between cell wall synthesis and hydrolysis, whichwould lead to cell wall thickening. The thickening of the cell walland its decreased cross-linking undoubtedly providemore free D-Ala–D-Ala residues. Thus, to arrive at the cell wall synthesis site,namely, the division septum, most of the vancomycin moleculemight be trapped, thereby elevating the vancomycin resistance inVISA. On the other hand, the alteration of central metabolism(TCA) might enhance the lipid cycle in VISA by regulating

the CcpE protein, which promotes the nascent peptidoglycanbiosynthesis, as well as the synthesis of LTA andWTA.Mutationsin metabolic genes, such as cmk, caused the downregulation ofLTA and WTA synthesis as well as increased the vancomycinresistance in VISA.

AUTHOR CONTRIBUTIONS

HP arranged the tables, QHwrote the manuscript, XR revised themanuscript.

FUNDING

This work was supported byNatural Science grants 81471993 and81672071 from National Natural Science Foundation of China.

ACKNOWLEDGMENTS

We thank Zhigang Huang for helping us in designingillustrations.

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Conflict of Interest Statement: The authors declare that the research was

conducted in the absence of any commercial or financial relationships that could

be construed as a potential conflict of interest.

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Frontiers in Microbiology | www.frontiersin.org 18 October 2016 | Volume 7 | Article 1601